Molecular Vision 2012; 18:1189-1196 Received 1 March 2012 | Accepted 3 May 2012 | Published 6 May 2012
© 2012 Molecular Vision
The preservative polyquaternium-1 increases cytoxicity and NFkappaB linked inflammation in human corneal epithelial cells Tuomas Paimela,1 Tuomas Ryhänen,1 Anu Kauppinen,1 Liisa Marttila,2 Antero Salminen,3,4 Kai Kaarniranta1,2 (The first two authors contributed equally to the work) 1Department
of Ophthalmology, Institute of Clinical Medicine, University of Eastern Finland, Kuopio, Finland; 2Department of Ophthalmology, Kuopio University Hospital, Kuopio, Finland; 3Department of Neurology, Institute of Clinical Medicine, University of Eastern Finland, Kuopio, Finland; 4Department of Neurology, Kuopio University Hospital, Kuopio, Finland Purpose: In numerous clinical and experimental studies, preservatives present in eye drops have had detrimental effects on ocular epithelial cells. The aim of this study was to compare the cytotoxic and inflammatory effects of the preservative polyquaternium-1 (PQ-1) containing Travatan (travoprost 0.004%) and Systane Ultra eye drops with benzalkonium chloride (BAK) alone or BAK-preserved Xalatan (0.005% latanoprost) eye drops in HCE-2 human corneal epithelial cell culture. Methods: HCE-2 cells were exposed to the commercial eye drops Travatan, Systane Ultra, Xalatan, and the preservative BAK. Cell viability was determined using colorimetric MTT (3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and by release of lactate dehydrogenase (LDH). Induction of apoptosis was measured with a using a colorimetric caspase-3 assay kit. DNA binding of the nuclear factor kappa B (NF-κB) transcription factor, and productions of the proinflammatory cytokines, interleukins IL-6 and IL-8, were determined using an enzyme-linked immunosorbent assay (ELISA) method. Results: Cell viability, as measured by the MTT assay, declined by up to 50% after exposure to Travatan or Systane Ultra solutions which contain 0.001% PQ-1. BAK at 0.02% rather than at 0.001% concentration evoked total cell death signs on HCE-2 cells. In addition, cell membrane permeability, as measured by LDH release, was elevated by sixfold with Travatan and by a maximum threefold with Systane Ultra. Interestingly, Travatan and Systane Ultra activated NF-κB and elevated the secretion of inflammation markers IL-6 by 3 to eightfold and IL-8 by 1.5 to 3.5 fold, respectively, as analyzed with ELISA. Conclusions: Eye drops containing PQ-1 evoke cytotoxicity and enhance the NF-κB driven inflammation reaction in cultured HCE-2 cells. Our results indicate that these harmful effects of ocular solutions preserved with PQ-1 should be further evaluated in vitro and in vivo.
Benzalkonium chloride (BAK) is the most commonly used preservative in ophthalmic drops. BAK has cytotoxic effects and it has been shown to induce inflammation on the ocular surface cells in numerous in vitro and in vivo models [1-11]. Since the clinical treatment of glaucoma or dry eye syndrome usually requires a long-term topical drug therapy, ocular side effects may be potentiated by the use of preserved ocular drops [4,6,7,12]. Polyquaternium-1 0.001% (Polyquad®, PQ-1) is a detergent-type preservative derived from BAK. PQ-1 was formulated in the mid of 1980s by Alcon as a preservative for contact lens storage solutions. Nowadays, it is being increasingly used as a preservative in ophthalmic drops for glaucoma and artificial tear solutions. Recent findings reveal that PQ-1 has detrimental effects on cell Correspondence to: Kai Kaarniranta, Department of Ophthalmology, University of Eastern Finland, P.O. Box 1627, 70211 Kuopio, Finland; Phone: +358-17-172485; FAX: +358-17-172486; email:
[email protected]
membrane integrity and it induces cytotoxicity in ocular surface cells [13,14]. Cell membrane damage may activate inflammation and cytotoxicity via Toll-like receptors (TLRs) that are a class of proteins playing a key role in the innate immune system [15]. TLRs are classical inducers of nuclear factor kappa B (NF-κB) transcription factor that is a ubiquitous inducible transcription factor, and the master regulator of acute and chronic immune responses, cellular proliferation and cell death. The NF-κB protein complex is maintained in the cytoplasm in an inactive state by the presence of inhibitory kappa proteins (IκBs). In response to various stress stimuli, IκB kinase (IKK) can phosphorylate IκB proteins which, in turn, leads to NF-κB activation through the formation of a heterodimer of p50 and p65 NF-κB subunits which is then translocated to the nucleus where it triggers the activation of many inflammatory genes, such as interleukins IL-6 and IL-8 [15]. This study explored the hypothesis that PQ-1 might have some detrimental effects on ocular surface cells. Therefore,
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cytotoxicity, cellular permeability and the inflammatory effects of commercial eye drops containing PQ-1 0.001% (Travatan and Systane Ultra) or an eye drop with BAK 0.02% (Xalatan) and pure BAK 0.001% or 0.02% were analyzed using HCE-2 human corneal epithelial cell cultures. METHODS Cell culturing and treatments: Cells used in this study were human corneal epithelial cells (HCE-2) obtained from American Type Culture Collection (ATCC, Manassas, VA). The cells were grown to confluency on 12-well plates (Cellstar®; Greiner Bio-One GmbH, Frickenhausen Germany) in a standard cell culture incubator (humidified CO2 10% athmosphere and 37 °C). Keratinocyte- Serum Free Medium (SFM, with bovine pituitary extract and epidermal growth factor, cat. no. 17005–042; Life Technologies, Invitrogen, GIBCO®, Paisley, UK) containing insulin 0.005 mg/ml (cat. no. I-6634; Sigma-Aldrich, Steinheim, Germany), fetal bovine serum 10% (FBS, cat. no. CH30160.03; Thermo Scientific, Hyclone, Logan, UT) and penicillin 100U/ml + streptomycin 100 µg/ml (cat. no. DE17–602E; Lonza, Basel, Switzerland) was used as the culture medium. Fresh medium was supplied to the cells every other day and the cells were subcultured twice a week using 0.25% trypsin-EDTA (cat. no. 25200056; Life Technologies, Invitrogen) to detach the cells from plates. The cells were exposed to the treatments (see below) for 5, 15, and 30 min, and then kept in the cell culture medium for 24 h, except for NF-κB activity test for 6 h, on 12 well plates before being analyzed. For every well 100,000 cells were seeded and cultured for 48 h before of exposures. Culture medium volume was 1 ml per dish, while for exposures used volume was 0.5 ml per dish. The cells were washed once with keratinocyte-SFM medium (without any supplements) before and after treatment to prevent any extra protein precipitation caused by BAK. The treatments were: 0-control (normal cell culture medium), Travatan (40 µg/ml travoprost, polyquaternium-1/polidronium chloride 0.001% as preservative; Alcon, Hünenberg, Switzerland), Systane Ultra (artificial tear drops, Polyquaternium-1/polidronium chloride 0.001% as preservative; Alcon), BAK 0.001% and 0.02% v/ v aqueous solution (FeF Chemicals A/S, Køge, Denmark), and Xalatan (0.005% latanoprost, BAK 0.02% as preservative; Pfizer, New York City, NY). Cell viability: MTT assay—The cytotoxicity of exposure was measured with MTT-assay [16]. Color of MTT tetrazole salt was measured with a spectrophotometer at the wavelength of 570 nm. Briefly, fresh MTT solution (10 mg/ml in 1× PBS) was added (1:20 volume of medium) and the cells were incubated for 1.5 h. The cells were lysed and purple formazan dissolved into the solution by overnight incubation with MTTlysis buffer (20% SDS, 50% N,N-dimethylformamide, 2%
© 2012 Molecular Vision
acetic acid, 25mM HCl; the volume of medium + volume of MTT-salt solution). LDH assay—The permeability of cellular membranes following the exposures was determined by measuring the amount of released lactate dehydrogenase (LDH) enzyme from HCE-2 cells. The commercial CytoTox 96® -kit (cat. no. G1780; Promega, Fitchburg, WI) was used according to the manufacturer’s instructions. Maximum LDH release of HCE-2 cells was determined by lysing HCE-2 cells for 45 min (lysis buffer provided within the assay), and subsequently measuring the LDH from the culture medium. Absorbance values after the colorimetric reaction were measured at the wavelength of 490 nm with a reference wavelength of 655 a BIO-RAD Model 550 microplate reader (BIO-RAD, Hercules, CA). Caspase-3—The levels of an apoptosis marker caspase-3 (active form) were measured from cell lysates using a colorimetric assay kit (cat. no. CASP-3-C; Sigma-Aldrich). Caspase-3 hydrolyses the peptide substrate Ac-DEVD-pNA (acetyl-Asp-Glu-Val-Asp p-nitroanilide) releasing pNA (pnitroaniline) which can be measured at the wavelength of 405 nm. The assay was performed according to the instructions of the manufacturer. Caspase-3 (Product Code C5974; Sigma) was used as a positive control, and the Assay Buffer provided with the kit served as a negative control.The absorbance values were measured using a BIO-RAD Model 550 microplate reader (BIO-RAD). Inflammation: IL-6 and IL-8 assays—The concentrations (pg/ml) of IL-6 were measured from cell culture medium samples in duplicates by BD OptEIA™ Human IL-6 ELISA Set (cat. no. 555220; BD Biosciences, San Diego, CA). The assay was performed according to the instructions of manufacturer. For determining the IL-8 concentrations (pg/ml), the BD OptEIA™ Human IL-8 ELISA Set (cat. no. 555244; BD Biosciences) was used. The absorbance values after the colorimetric reaction were measured at the wavelength of 450 nm with a reference wavelength of 655 nm using a BIO-RAD Model 550 microplate reader (BIO-RAD). NF-κB assay—NF-κB p65 ELISA kit (cat. no. EKS-446) was obtained from Enzo Life Sciences (Lausen, Switzerland) and used to measure p65 subunit after the binding to DNA. The cells were lysed in 25% glycerol, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 20 mM HEPES before analyses, and the assay was performed according to the manufacturer’s instructions using 10 μg of protein per well. The luminescence signal was measured using VICTOR™ 1420 multilabel counter (PerkinElmer/Wallac, Turku, Finland). Statistical analysis: Statistical analyses were conducted with GraphPad Prism (Graphpad Software, San Diego, CA). Differences between groups were analyzed with the one-way ANOVA test followed by Dunnett’s post hoc tests. P-values
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© 2012 Molecular Vision
Figure 1. Level of cytotoxicity in HCE-2 cells analyzed by MTT assay. Columns represent the viability of cells (mean±SD. The viability of control cells is set as 100%. One-Way ANOVA, followed by Dunnett’s post hoc test, evaluated the statistical differences (n=6, *0.01
